Husker team wins prize in contest to treat disease through gene editing

19 Feb 2024     By Tiffany Lee, Research and Economic Development

A Husker research duo was named a first-round winner in a National Institutes of Health competition aimed at generating solutions for delivering genome-editing technology to the cells of people with rare and common diseases.

Janos Zempleni, Willa Cather Professor of molecular nutrition, and Jiantao Guo, professor of chemistry, were selected as Phase 1 winners in the NIH’s Targeted Genome Editor Delivery Challenge. The challenge is a three-phase competition with prizes totaling $6 million; the University of Nebraska–Lincoln team was among 30 initial recipients announced in December 2023.

With the $25,000 prize, Zempleni and Guo will advance development of universal milk exosomes — natural nanoparticles contained in milk — capable of transporting gene editors to any location in the body.

These programmable exosomes would be safe, scalable and, unlike conventional nanoparticles, capable of evading macrophages, the immune system cells that destroy foreign substances.

“With our technology, you could treat basically any disease known to mankind, both rare and common,” said Zempleni, who is leading the project and directs the Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules. “Federal agencies are not overly eager to invest in treatment of rare diseases because it costs a lot of money, yet only a few people benefit.

“With the flexibility of our technology, we can, without extra cost, provide a platform to treat everything, from common diseases like brain cancer, to very rare gene mutations that maybe affect only 500 people in the United States.”

The technology would overcome one of the most significant challenges to using gene editing to treat disease. Though tools like CRISPR-cas9 can delete, repair or replace disease-causing DNA — which could halt tumor growth in cancer or shut down production of harmful proteins — there is currently no reliable means of guaranteeing an editor will reach whichever organ or tissues are associated with a certain disease. And, when gene editors do reach the intended target, they often do not survive in sufficient numbers to change the course of the disease.

The Husker team’s solution combines Zempleni’s nationally recognized expertise in milk exosomes with Guo’s extensive skills in the growing field of bioorthogonal chemistry. Based on his previous research, Zempleni knew it was possible to load milk exosomes with therapeutics and deploy them to a specific tissue in the body. He’s also demonstrated the exosomes’ biological safety, meaning they’re unlikely to cause adverse reactions in patients.

But his tools lacked versatility: He needed a method for directing the exosomes to different locations depending on the disease at hand.

To accomplish this, Zempleni developed an approach that allows him to attach three peptides — short amino acid chains — to the membrane of each exosome. One is a homing peptide, which directs the exosome to bind to a specific site in the body. Another is a “do not eat me” peptide, which sends biochemical signals that allow the exosome to thwart macrophage destruction. The last is what is called a retrofusion peptide, which fortifies the exosome’s survivability once it enters the target cells.

Using the three peptides in concert is novel, Zempleni said, and preliminary data indicate the approach is feasible. But what iss even more innovative is the team’s approach for anchoring the peptides to the exosome membrane. Conventional approaches insert lipid anchors into the membrane, then attach the peptide modifiers to those anchors. The problem is that those lipids are attracted to other lipophilic compounds in the body, leading to detachment and loss of the attached peptide.

The Nebraska team’s approach overcomes this problem by creating docking sites in a membrane protein called CD81, which is firmly rooted in the exosome, preventing detachment. Guo is using bioorthogonal chemistry approaches to create stable, covalent links between the docking sites and the peptides. He said this attachment scheme confers stability and uniformity to the exosome structure, boosting the commercial viability of milk exosome-based therapeutics.

“If this were to go to clinical trials, it will be easier for the FDA to see that this is a defined, homogenous structure, rather than random labeling that would lead to batch-to-batch differences,” said Guo, who leads the Nebraska Center for Integrated Biomolecular Communication.

Zempleni and Guo are also attaching a polyhistidine tag to the exosomes, which allows them to be purified at a low cost and a high purity. The tag is easily removable in case it causes adverse reactions in patients.

Packaging the milk exosomes with CRISPR-cas9 cargo and other therapeutics is one of Zempleni’s longer-term goals. Eventually — possibly with support from additional funding through the TARGETED Challenge — he aims to partner with a transgenic livestock expert at Utah State University to develop a goat or cow that, through its milk, secretes massive numbers of programmable exosomes containing gene-editing cargo and the peptide docking sites.

For this Phase 1 project, he will load cultured MAC-T cells, which closely mirror cow milk cells, with gene-editing tools using previously established genetics and chemistry approaches.

Zempleni and Guo’s designer exosomes are already showing commercial potential. At the end of 2023, the team was notified they would receive a patent titled Extracellular Vesicles and Methods of Using. They have licensed the inventions in that patent to a private company, which is partnering with Roche, Inc., to use bovine milk exosome technology to deliver RNA therapeutics to brain tumors.

The researchers are preparing their application for the TARGETED Challenge Phase 2, which will award up to 10 winners $250,000 and eligibility to compete in Phase 3. NIH will announce Phase 2 winners in April 2025.